Peptides containing the motif IGD and their use as cell migration modulators

ABSTRACT

A compound with a relative molecular mass of less than 15000 comprising the peptide Ile-Gly-Asp (IGD) or a peptide or non-peptide mimic thereof. The compounds may be used to modulate cell migration and are useful in angiogenesis.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to peptides and related compounds and inparticular to peptides and related compounds which affect cellmigration.

2. Description of the Related Art

Fibronectin is a widely distributed glycoprotein present at highconcentrations in most extracellular matrices, in plasma (300 μg/ml),and in other body fluids. Fibronectin is a prominent adhesive proteinand mediates various aspects of cellular interactions with extracellularmatrices including migration. Its principal functions appear to be incellular migration during development and wound healing, regulation ofcell growth and differentiation, and haemostasis/thrombosis.

Fibronectin is a dimer of two non-identical subunits covalently linkednear their COOH-termini by a pair of disulphide bonds. The differencebetween the subunits is determined by alternative splicing of the IIICS(or V) region. In the insoluble, matrix form of fibronectin, the dimerassociates into disulphide-bonded oligomers and fibrils, while soluble,body fluid fibronectin is predominantly dimeric. Three regions offibronectin are subject to alternative splicing and in general thematrix form of the molecule has a higher content of these segments thanthe soluble form. The human IIICS region has five potential variations,while the rat, bovine and chicken sequences have three, three and two,respectively. Each subunit is composed of a series of structurallyindependent domains linked by flexible polypeptide segments. At theprimary sequence level, the origin of the majority of the fibronectinmolecule can be accounted for by endoduplication of three types ofpolypeptide repeat. Different fibronectin domains are specialized forbinding extracellular matrix macromolecules or bacterial or eukaryoticmembrane receptors. The central cell-binding domain is recognised bymost adherent cells via the integrin receptors α3β1, α5β1, αVβ1, αllbβ3,αVβ3, αVβ5 and αVβ6. The IIICS/Hepll cell-binding domain is recognisedby lymphoid cells, neural crest derivatives and myoblasts via theintegrins α4β1 and α4β7. Several peptide active sites have beenidentified in these domains.

Plasma fibronectin can be purified by a combination of gelatin andheparin affinity chromatography. Cell-associated fibronectin can beextracted from culture monolayers with 1 M urea. Further details onfibronectin are in The Extracellular Matrix Facts Book, Ayad et al(eds), Academic Press, Harcourt Brace & Company, London.

Limited proteolytic digestion of fibronectin results in the release of anumber of its functional domains, which are characterised by theirspecific adhesion to other matrix macromolecules or integrin receptorson the cell surface (i.e. the cell-binding domain)¹. The transmembraneassay has commonly been used to study the effects of fibronectin and itspurified functional domains on cell migration in vitro. Essentially,this assay involves assessing cell movement through a polycarbonatemembrane coated with an adhesive protein (usually gelatin) separatingupper and lower medium compartments containing the putative effectormolecule. Previous studies have revealed that nano- to micromolarconcentrations of fibronectin and its purified cell-binding domainstimulate the migration of a wide range of cell types in thetransmembrane assay^(2,3). Related studies implicated the RGD (SEQ ID NO4) amino acid motif (located in the tenth type III repeat module) inmediating these effects of both native fibronectin and its cell-bindingdomain³. Significantly, small RGD-containing synthetic peptides did notstimulate cell migration; indeed, these peptides inhibited the adhesiveand migration stimulating activity of larger protein domains containingthe RGD (SEQ ID NO 4) motif by competition for receptor ligation⁴. Incontrast to the activity of the cell-binding domain, the gelatin-bindingdomain of fibronectin has consistently been reported to be devoid ofmigration stimulating activity in the transmembrane assay^(2,5).

Schor et al (1994) Progress in Growth Factor Research 5, 223–248 is areview of cytokine control of cell motility and its modulation andmediation by the extracellular matrix. Schor et al (1993) In: CellBehaviour: Adhesion and Motility (ed. G. Evans, C. Wigley and R. Warn)Society of Experimental Biology Symposium No. 47, pages 235–251 relatesto migration stimulating factor (MSF).

Grey et al (1989) Proc. Natl. Acad. Sci. USA 86, 2438–2442 relates tothe purification of the MSF produced by fetal and breast cancer patientfibroblasts.

U.S. Pat. No. 5,300,630 relates to oncodevelopmentally regulatedantigens related to fibronectin.

U.S. Pat. No. 5,510,328 relates to methods of reducing or inhibitingwound contraction using certain peptides.

U.S. Pat. No. 5,354,736 relates to compounds that have enhanced cellbinding with respect to collagen.

U.S. Pat. No. 4,976,734 relates to a method of stimulating chemotaxistowards a prosthetic device.

U.S. Pat. No. 4,980,279 relates to portions of fibronectin.

U.S. Pat. No. 5,049,658 relates to a polypeptide having thecell-spreading activity of human fibronectin.

U.S. Pat. No. 5,124,155 relates to wound healing dressings, which areprepared by flocculating fibronectin.

U.S. Pat. No. 5,453,489 relates to polypeptides, which encompassfibronectin—fibronectin binding sites and which are capable ofinhibiting fibronectin matrix assembly.

U.S. Pat. No. 5,192,746 relates to compounds having the property ofmodulating cell adhesion.

U.S. Pat. No. 5,491,130 relates to peptides derived from humanendothelial cell thrombosponding, which bind to the gelatin-bindingdomain of fibronectin.

SUMMARY OF THE INVENTION

We have previously developed an alternative migration assay involvingthe assessment of cell movement into gels of native type I collagenfibres⁶. Using this assay, we have recently reported that femtomolarconcentrations of the gelatin-binding domain stimulated the migration ofhuman dermal fibroblasts, whilst native fibronectin and its cell-bindingdomain were inactive⁷.

We observed that the gelatin-binding domain did indeed stimulate cellmigration in the transmembrane assay when filters were coated withnative collagen, but not with gelatin (as used in the majority ofprevious studies). One of the objectives of the present study has beento determine whether a candidate amino acid sequence within thegelatin-binding domain of fibronectin is responsible for itssubstratum-dependent stimulation of cell migration and, if so, themanner in which it may be functionally related to the RGD (SEQ ID NO 4)motif. Surprisingly we have found that peptides and other moleculescontaining the IGD (SEQ ID NO 1) amino acid sequence motif stimulatefibroblast migration into native but not denatured collagen substrate.

A first aspect of the invention provides a compound with a relativemolecular 20 mass of less than 15 000 comprising the peptide Ile-Gly-Asp(IGD) (SEQ ID NO 1) or a peptide or non-peptide mimic thereof.

Preferably, the compound has a relative molecular mass of less than12000, more preferably less than 10000.

We have found that, surprisingly, the peptide IGD (SEQ ID NO 1) alone orwhen present as a moiety in a larger molecule is able to modulate cellmigration. Thus, the preferred compounds of the invention are those,which are able to modulate cell migration under appropriate conditionssuch as those conditions described in the Examples.

By peptide mimic of IGD (SEQ ID NO 1) we include that the Ile isreplaced by another hydrophobic amino acid such as Val, Leu, Phe, Trp orTyr, most preferably Val or Leu. We also include that the Asp isreplaced by Glu. Less preferably the Gly residue is replaced by Ala. Thepeptide mimics are preferably those that exhibit substantially the samecell migration modulating activity of a peptide comprising the peptidesequence IGD (SEQ ID NO 1) and, more preferably substantially the samecell migration modulating activity of any one of the peptides IGD (SEQID NO 1) or IGDS (SEQ ID NO 2) or IGDQ (SEQ ID NO 3). Suitably, thepeptide mimic comprises a moiety, which has substantially the samecharge distribution and/or spatial configuration as any one of thepeptides IGD (SEQ ID NO 1) or IGDS (SEQ ID NO 2) or IGDQ (SEQ ID NO 3).

By non-peptide mimics of IGD (SEQ ID NO 1) we include a moiety which hasthe same charge distribution and/or spatial configuration as any one ofthe peptides IGD (SEQ ID NO 1) or IGDS (SEQ ID NO 2) or IGDQ (SEQ ID NO3), and we include a moiety which has substantially the same cellmigration modulating activity of a peptide comprising the IGD (SEQ IDNO 1) sequence.

Non-peptide surrogates of the RGD (SEQ ID NO 4) sequence have beendeveloped or are described by, for example, Greenspoon et al (1993)Biochemistry 32, 1001–1008 and Humphries et al (1994) Exp. Opin. Ther.U.S. Pat. No. 4, 227–235, both of which are incorporated herein byreference, and suitable non-peptide mimics based on the IGD (SEQ IDNO 1) motif can be made using similar principles as for the non-peptidemimics of RGD. It can be well appreciated that I can be substituted forR and that the resulting peptides can be tested for cell increasingmigration activity using the techniques described herein.

In Greenspoon et al., the design and preparation of nonpeptide analoguesof RGD are described as follows. Compounds SF-6,5 and SFN-70 wereprepared by coupling of methyl-5-aminovaleric acid withN-(butyloxycarbonyl)-6-aminohexanoic acid. The reaction was carried outusing the 1,3-dicyclohexylcarbodiimide and 1 hydroxybenzotriazole intetrahydroduran procedure. The butyloxycarbonyl protecting group wasthen removed by 50% trifluoroacetic acid in dichloromethane. Removal ofthe methyl ester protecting group was carried out with sodium hydroxideat pH 9.5, yielding SFN-70. The amine was converted to guanidium using3,5-dimethylpyrazole-1-carboxamidine nitrate at pH 9.5 t produce thecompound designated SF-6,5. Compound SF-6,6 was prepared frommethyl-6-aminohexanoic acid and N-(butyloxycarbonyl)-6-aminohexanoicacid under the same conditions as used for SF-6,5. GK-5,5 was preparedfrom methyl-5-aminovaleric acid and N-(butyloxycarbonyl)-5-aminovalericacid. Compounds AC-4 and AC-14 were prepared by stepwise synthesiseither in solution or on a Merrifield resin, followed by deprotectionand conversion of the amine to the guanidinium by the above-describedmethods. The final compounds were purified on preparative RP-18 columnsand were judged pure by thin-layer chromatography (single spot) and ¹HNMR spectroscopy. Compounds were characterized by ¹H NMR and FAB-MSspectroscopy. The structures deduced from spectroscopy were consistentwith the assigned structure. (Table 1). Table 1 shows a schematicpresentation of the RGD-NH₂ peptide and the corresponding non-peptidesurrogates. Note that the distance between positions a and b is 11atoms.

TABLE 1

No. of atoms in the Compound spacer SF-6,5

11 AC-4

11 AC-14

11 SF-6,6

12 SFN-70

11 GK-5,5

10

In Humphries et al., the design of nonpeptide analogues of RGD aredescribed as follows. Complete deletion of the acidic functionality istolerated in special circumstances, as with the antiparasitic drug,pentamidine, which possesses two amidinium groups and can perturb αIIbβ3function. Simple des-aspartyl basic motifs have also been selected inpeptide library panning studies with αIIbβ3. The backbone scaffold towhich these key functions are attached can be simplified and varied inremarkable fashion. The more potent antagonists tend to replicate thehydrophilic character of the native peptide backbone to some degree,although there is no obvious requirement for hydrogen bonding to thereceptor. Indeed, simple flexible constructs or constrained hydrocarbonscaffolds still possess marked affinity. Table 2 shows a survey ofsynthetic RGD analogues.

TABLE 2 Compound Source Reference

Smith Kline &French J. Am. Chem.Soc. (1992) 114:9615–9623

Merck J. Med. Chem.(1992) 35, 4640–4642

Glaxo Glaxo, EP-542363-A

Hoffman-LaRoche Hoffman-LaRoche, EP-372486-A2

Dr. Karl Thomae Dr. Karl Thomae,EP-483667-A2

GD Searle GD Searle, EP-502536-A1

[Pentamidine] Thromb.Haemostasis(1992) 68(6):731–736

Greenspoon et al. Biochemistry(1993) 32:1001–1008

Hirschmann et al. Tetrahedron(1993)49(17):3665–3676

Compounds which exhibit substantially the same cell migration modulatingactivity of a peptide comprising the peptide Ile-Gly-Asp (IGD) (SEQ IDNO 1) can be selected using a suitable screening system. The preferredscreening system uses a migration assay similar to that described inExample 1 in which cell migration is assessed on a native type Icollagen substratum (for example, collagen gel or transmembrane assayusing collagen-coated membranes). Mimics are compounds which exhibitsubstantially the same effect as the peptides IGD (SEQ ID NO 1) or IGDS(SEQ ID NO 2) or IGDQ (SEQ ID NO 3) and which may act in an additivefashion with them. Inhibitors would abrogate the bioactivity of thepeptides IGD (SEQ ID NO 1) or IGDS (SEQ ID NO 2) or IGDQ (SEQ ID NO 3).The peptide IGD (SEQ ID NO 1) and the gel-binding domain (GBD) act in anadditive fashion, whereas the RGD (SEQ ID NO 4) peptide is an inhibitorof the IGD (SEQ ID NO 1) peptide.

Although the peptide sequence IGD (SEQ ID NO 1), when present in acompound of the invention, is sufficient to modulate cell migration itis preferred if the peptide sequence Ile-Gly-Asp-Ser (IGDS) orIle-Gly-Asp-Gin (IGDQ)(SEQ ID NO 3) is present in a compound of theinvention.

We have found, in relation to the peptides IGDS (SEQ ID NO 2), and IGD(SEQ ID NO 1) that is more potent than IGDQ (SEQ ID NO 3) which is morepotent than IGD (SEQ ID NO 1) in stimulating fibroblast migration and soit is preferred if the compound comprises the peptide IGDS (SEQ ID NO2).

It is preferred that the amino acids within the peptide sequence IGD(SEQ ID NO 1) or IGDS (SEQ ID NO 2) or IGDQ (SEQ ID NO 3), when presentin the compounds of the invention, are all in the L configuration as isthe case for natural amino acids. It will, nevertheless, be appreciatedthat when the compound of the invention is a peptide comprising IGD (SEQID NO 1) or IGDS (SEQ ID NO 2) or then the other amino acids within thepeptide (ie those other than the ones in the IGD (SEQ ID NO 1) or IGDS(SEQ ID NO 2) or IGDQ (SEQ ID NO 3) moiety may be in the L- orD-configurations. In as much as peptides containing D-amino acids may bemore resistant to proteolysis compounds containing D-amino acids (otherthan in the IGD (SEQ ID NO 1) or IGDS (SEQ ID NO 2) or IGDQ (SEQ ID NO3) moiety may be preferred.

The invention covers all compounds with a relative molecular mass ofless than 15 000 comprising the peptide IGD (SEQ ID NO 1) or a peptideor non-peptide mimic thereof; however, it is particularly preferred ifthe compound is a peptide comprising the peptide moiety IGD (SEQ ID NO1). In other words, preferred compounds are peptides larger than 3 aminoacids which contain the peptide moiety IGD (SEQ ID NO 1). Such peptidesinclude the peptides IGDS (SEQ ID NO 2) and IGDQ (SEQ ID NO 3) and alsoinclude peptides with additional amino acids N terminal and/or Cterminal to these motifs.

The compounds of the invention also include peptides wherein the IGD(SEQ ID NO 1) or IGDS (SEQ ID NO 2) or IGDQ (SEQ ID NO 3) moiety ismasked by, for example, blocking groups being present on free —NH2 or—COOH groups. Preferably, such blocking groups are ones which may beremoved readily in vivo, for example by hydrolysis; however, in somecircumstances it may be desirable if the blocking groups aresubstantially resistant to hydrolysis.

The peptide IGD (SEQ ID NO 1) is also a compound of the invention.

It is preferred if the compounds have a relative molecular mass of lessthan 8000, preferably less than 6000, more preferably less than 5000,and preferably less than 2000. When the compound of the invention is apeptide it is preferred if the peptide has between 4 and 80 amino acidresidues, more preferably between 4 and 50, still more preferablybetween 4 and 30 and preferably between 4 and 20 amino acid residues.

The compound of the invention may have a linear configuration or it maybe branched or circular. When the compound of the invention is a peptideit may be linear, branched or circular.

It will be appreciated that the compound of the invention may comprisemore than one IGD (SEQ ID NO 1) peptide moiety or a peptide ornon-peptide mimic thereof. In certain circumstances it is advantageousfor the compound of the invention to comprise between two and 50 suchpeptide moieties or a peptide or non-peptide mimics thereof, morepreferably between 2 and 20 and most preferably between 5 and 15 suchpeptide moieties or peptide or non-peptide mimics thereof.

Thus, the peptide of the invention may consist of multiple repeats ofIGD (SEQ ID NO 1) or IGDS (SEQ ID NO 2) or IGDQ (SEQ ID NO 3) or apeptide sequence containing conservative substitutions for I, G or D(such as IGES (SEQ ID NO 5)), and it may consist of any of these,including multiple repeats, in a cyclic form.

When the compound of the invention is a peptide it is possible for it tocontain tandem repeats of the IGD-containing moiety (such as IGD (SEQ IDNO 1) itself or IGDS (SEQ ID NO 2) or IGDQ (SEQ ID NO 3) or combinationsthereof).

Although not essential, it is preferred if the peptide of the inventioncomprises the IGD (SEQ ID NO 1) motif and flanking regions from thefibronectin molecule. For example, the flanking regions may be 1, 2, 3,4, 5 or more amino acid residues on one or both sides of the occurrenceof IGD (SEQ ID NO 1) in the fibronectin molecule. The amino acidsequence of the human fibronectin molecule is given in FIG. 5. Peptidescontaining flanking regions of fibronectin may have greater bioactivitythan shorter tri- and tetrapeptides. These flanking regions arepreferably derived from the seventh (for IGDQ (SEQ ID NO 3)) and ninth(for IGDS (SEQ ID NO 2)) type I repeat modules for fibronectin. It isalso preferred if the compounds of the invention are the intactfibronectin type I repeat modules (17 and 19) since these may exhibithigher bioactivity than shorter synthetic peptides. Each of these type Imodules contains approximately 45 amino acids and, preferably up tothree of these modules are used in tandem array.

When the compound of the invention is a peptide it may be synthesisedusing well-known methods in the art. For example, peptides may besynthesised by the Fmoc-polyamide mode of solid-phase peptide synthesisas disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and referencestherein. Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is effected using 20% piperidine inN,N-dimethylformamide. Sidechain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalisingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversedN,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used are ethanedithiol, phenol, anisole and water,the exact choice depending on the constituent amino acids of the peptidebeing synthesised. Trifluoroacetic acid is removed by evaporation invacuo, with subsequent trituration with diethyl ether affording thecrude peptide. Any scavengers present are removed by a simple extractionprocedure which on lyophilisation of the aqueous phase affords the crudepeptide free of scavengers. Reagents for peptide synthesis are generallyavailable from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.Purification may be effected by any one, or a combination of, techniquessuch as size exclusion chromatography, ion-exchange chromatography and(principally) reverse-phase high performance liquid chromatography.Analysis of peptides may be carried out using thin layer chromatography,reverse-phase high performance liquid chromatography, amino-acidanalysis after acid hydrolysis and by fast atom bombardment (FAB) massspectrometric analysis.

Alternatively, when the peptide of the invention is of a suitable size,such as greater than about 50 residues in length, it may be desirable toproduce the peptide by recombinant DNA technology.

The peptides of the invention may be encoded by a suitablepolynucleotide which may be obtained or synthesised by methods wellknown in the art.

The DNA is then expressed in a suitable host to produce a peptidecomprising the compound of the invention. Thus, the DNA encoding thepeptide constituting the compound of the invention may be used inaccordance with known techniques, appropriately modified in view of theteachings contained herein, to construct an expression vector, which isthen used to transform an appropriate host cell for the expression andproduction of the peptide of the invention. Such techniques includethose disclosed in U.S. Pat. No. 4,440,859 issued 3 Apr. 1984 to Rutteret al, U.S. Pat. No. 4,530,901 issued 23 Jul. 1985 to Weissman, U.S.Pat. No. 4,582,800 issued 15 Apr. 1986 to Crowl, U.S. Pat. No. 4,677,063issued 30 Jun. 1987 to Mark et al, U.S. Pat. No. 4,678,751 issued 7 Jul.1987 to Goeddel, U.S. Pat. No. 4,704,362 issued 3 Nov. 1987 to Itakuraet al, U.S. Pat. No. 4,710,463 issued 1 Dec. 1987 to Murray, U.S. Pat.No. 4,757,006 issued 12 Jul. 1988 to Toole, Jr. et al, U.S. Pat. No.4,766,075 issued 23 Aug. 1988 to Goeddel et al and U.S. Pat. No.4,810,648 issued 7 Mar. 1989 to Stalker, all of which are incorporatedherein by reference.

The DNA encoding the peptide constituting the compound of the inventionmay be joined to a wide variety of other DNA sequences for introductioninto an appropriate host. The companion DNA will depend upon the natureof the host, the manner of the introduction of the DNA into the host,and whether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognised bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance. Alternatively, the gene for such selectable traitcan be on another vector, which is used to co-transform the desired hostcell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the peptide, which can thenbe recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus), plant cells,animal cells and insect cells.

The vectors include a prokaryotic replicon, such as the ColEl ori, forpropagation in a prokaryote, even if the vector is to be used forexpression in other, non-prokaryotic, cell types. The vectors can alsoinclude an appropriate promoter such as a prokaryotic promoter capableof directing the expression (transcription and translation) of the genesin a bacterial host cell, such as E. coli, transformed therewith.

A promoter is an expression control element formed by a DNA sequencethat permits binding of RNA polymerase and transcription to occur.Promoter sequences compatible with exemplary bacterial hosts aretypically provided in plasmid vectors containing convenient restrictionsites for insertion of a DNA segment of the present invention.

Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99Aand pKK223-3 available from Pharmacia, Piscataway, N.J., USA.

A typical mammalian cell vector plasmid is pSVL available fromPharmacia, Piscataway, N.J., USA. This vector uses the SV40 latepromoter to drive expression of cloned genes, the highest level ofexpression being found in T antigen-producing cells, such as COS-1cells.

An example of an inducible mammalian expression vector is pMSG, alsoavailable from Pharmacia. This vector uses the glucocorticoid-induciblepromoter of the mouse mammary tumour virus long terminal repeat to driveexpression of the cloned gene.

Useful yeast plasmid vectors are pRS403–406 and pRS413–416 and aregenerally available from Stratagene Cloning Systems, La Jolla, Calif.92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (Yips) and incorporate the yeast selectable markersHIS3, TRP1, LEU2 and URA3. Plasmids pRS413–416 are Yeast Centromereplasmids (YCps).

A variety of methods have been developed to operably link DNA to vectorsvia complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the 5 complementary homopolymeric tails to formrecombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion as describedearlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNApolymerase I, enzymes that remove protruding, 3′-single-stranded terminiwith their 3′–5′-exonucleolytic activities, and fill in recessed 3′-endswith their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNAsegments. The blunt-ended segments are then incubated with a large molarexcess of linker molecules in the presence of an enzyme that is able tocatalyze the ligation of blunt-ended DNA molecules, such asbacteriophage T4 DNA ligase. Thus, the products of the reaction are DNAsegments carrying polymeric linker sequences at their ends. These DNAsegments are then cleaved with the appropriate restriction enzyme andligated to an expression vector that has been cleaved with an enzymethat produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies Inc, New Haven, Conn., USA.

A desirable way to modify the DNA encoding the polypeptide of theinvention is to use the polymerase chain reaction as disclosed by Saikiet al (1988) Science 239, 487–491.

In this method the DNA to be enzymatically amplified is flanked by twospecific oligonucleotide primers which themselves become incorporatedinto the amplified DNA. The said specific primers may containrestriction endonuclease recognition sites which can be used for cloninginto expression vectors using methods known in the art.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells are preferredprokaryotic host cells and typically are a strain of E. coli such as,for example, the E. coli strains DH5 available from Bethesda ResearchLaboratories Inc., Bethesda, Md., USA, and RR1 available from theAmerican Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC31343). Preferred eukaryotic host cells include yeast and mammaliancells, preferably vertebrate cells such as those from a mouse, rat,monkey or human fibroblastic cell line. Yeast host cells include YPH499,YPHSO0 and YPHSO1 which are generally available from Stratagene Cloning.Systems, La Jolla, Calif. 92037, USA. Preferred mammalian host cellsinclude Chinese hamster ovary (CHO) cells available from the ATCC asCCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC asCRL 1658, and monkey kidney-derived COS-1 cells available from the ATCCas CRL 1650.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl.Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Transformation of yeast cells is described in Sherman et al (1986)Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y.The method of Beggs (1978) Nature 275, 104–109 is also useful. Withregard to vertebrate cells, reagents useful in transfecting such cells,for example calcium phosphate and DEAE-dextran or liposome formulations,are available from Stratagene Cloning Systems, or Life TechnologiesInc., Gaithersburg, Md. 20877, USA.

Electroporation is also useful for transforming cells and is well knownin the art for transforming yeast cell, bacterial cells and vertebratecells.

For example, many bacterial species may be transformed by the methodsdescribed in Luchansky et al (1988) Mol. Microbiol. 2, 637–646incorporated herein by reference. The greatest number of transformantsis consistently recovered following electroporation of the DNA-cellmixture suspended in 2.5× PEB using 6250V per cm at 25 μFD.

Methods for transformation of yeast by electroporation are disclosed inBecker & Guarente (1990) Methods Enzymol. 194, 182.

Successfully transformed cells, ie cells that contain a DNA construct ofthe present invention, can be identified by well-known techniques. Forexample, cells resulting from the introduction of an expressionconstruct of the present invention can be grown to produce thepolypeptide of the invention. Cells can be harvested and lysed and theirDNA content examined for the presence of the DNA using a method such asthat described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al(1985) Biotech. 3, 208. Alternatively, the presence of the protein inthe supernatant can be detected using antibodies as described below.

In addition to directly assaying for the presence of recombinant DNA,successful transformation can be confirmed by well-known immunologicalmethods when the recombinant DNA is capable of directing the expressionof the protein. For example, cells successfully transformed with anexpression vector produce proteins displaying appropriate antigenicity.Samples of cells suspected of being transformed are harvested andassayed for the protein using suitable antibodies. Thus, in addition tothe transformed host cells themselves, the present invention alsocontemplates a culture of those cells, preferably a monoclonal (clonallyhomogeneous) culture, or a culture derived from a monoclonal culture, ina nutrient medium.

Thus, a second aspect of the invention provides a polynucleotideencoding a peptide of the invention.

A third aspect of the invention provides a vector comprising apolynucleotide of the invention and a fourth aspect of the inventionprovides a host cell comprising a polynucleotide or vector of theinvention.

The compounds of the invention are useful in modulating cell migrationand therefore are useful in medicine.

Thus, a fifth aspect of the invention provides a compound according tothe first aspect of the invention for use in medicine.

A sixth aspect of the invention provides a pharmaceutical compositioncomprising a compound according to the first aspect of the invention anda pharmaceutically acceptable carrier.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the activeingredient (compound of the invention) with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surface-active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to provideslow or controlled release of the active ingredient therein using, forexample, hydroxypropylmethylcellulose in varying proportions to providedesired release profile.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of an activeingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

It is particularly preferred if the formulation is for topicaladministration, for example to the site of a wound.

It will be appreciated that some of the compounds of the invention willbe in the form of salts.

Salts which may be conveniently used in therapy include physiologicallyacceptable base salts, for example, derived from an appropriate base,such as an alkali metal (e.g. sodium), alkaline earth metal (e.g.magnesium) salts, ammonium and NX₄ ⁺ (wherein X is C₁₋₄ alkyl) salts.Physiologically acceptable acid salts include hydrochloride, sulphate,mesylate, besylate, phosphate and glutamate.

Salts according to the invention may be prepared in conventional manner,for example by reaction of the parent compound with an appropriate baseto form the corresponding base salt, or with an appropriate acid to formthe corresponding acid salt.

The aforementioned compounds of the invention or a formulation thereofmay be administered by any conventional method including oral andparenteral (e.g. subcutaneous or intramuscular) injection. The treatmentmay consist of a single dose or a plurality of doses over a period oftime.

Whilst it is possible for a compound of the invention to be administeredalone, it is preferable to present it as a pharmaceutical formulation,together with one or more acceptable carriers. The carrier(s) must beacceptable in the sense of being compatible with the compound of theinvention and not deleterious to the recipients thereof. Typically, thecarriers will be water or saline which will be sterile and pyrogen free.

A further aspect of the invention provides a method of modulating cellmigration the method comprising administering an effective amount of acompound according to the first aspect of the invention to the sitewhere modulation of cell migration is required.

Preferred compounds are those preferred in the first aspect of theinvention.

Cell migration may be modulated according to the method of this aspectof the invention in vitro, for example in cell culture systems, or itmay be modulated in vivo.

Impaired cell migration is commonly a feature of clinical conditions inwhich wound healing is not optimal; the stimulation of cell migrationunder these conditions may prove beneficial. Conversely, elevated orinappropriate cell migration is a feature of several pathologicalconditions, including tumour invasion, pathological angiogenesis,inflammation and fibrosis. Inhibitors of IGD (SEQ ID NO 1) bioactivitymay prove useful in the treatment of these conditions. Inhibitors of IGD(SEQ ID NO 1) bioactivity may be screened for using method apparent tothe skilled person based on the information contained herein.

The modulation of cell migration is desirable in, for example, woundhealing, guided periodontal tissue regeneration, inhibition of tumourinvasion and metastasis, and the compounds of the invention are alsouseful because of their effects on angiogenesis (new blood vesselformation). The compounds of the invention may also be useful inrelation to inflammation or connective tissue function.

Thus, it is preferred that the site where modulation of cell migrationis in an animal body, for example a mammalian, especially human, body.It is also preferred if the cell whose migration is modulated is afibroblast cell. We have shown that vascular cells are responsive tocertain compounds of the invention (for example, IGDS), and that IGDS(SEQ ID NO 2) stimulates angiogenesis in the chick yolk sac assay. Thus,the compounds of the invention are believed to be clinically useful instimulating angiogenesis in conditions such as impaired wound healing.

It will be seen, therefore, that the invention includes a method oftreating an animal, for example a mammal, especially human, in need ofmodulation of cell migration the method comprising administering to theanimal an effective amount of a compound according to the first aspectof the invention.

A further aspect of the invention provides the use of a compound of theinvention for modulating cell migration, especially in wound healing orperiodontal tissue regeneration or inhibition of tumour invasion andmetastasis or in modulating angiogenesis.

A still further aspect of the invention therefore provides use of acompound of the invention in the manufacture of a medicament formodulating cell migration in an animal body.

BRIEF DESCRIPTION OF THE DRAWINGS

Detailed description of the preferred embodiment of the invention willbe made with reference to the accompanying drawings.

FIGS. 1A (top) and 1B (bottom) are graphs showing the effects ofsynthetic peptides and fibronectin domains on cell migration in thenative collagen gel assay;

FIGS. 2A (top) and 2B (bottom) are graphs showing the effects ofsynthetic peptides and fibronectin domains on cell migration in thetransmembrane assay;

FIGS. 3A (top), 3B (middle) and 3C (bottom) are graphs showing theeffects of different peptides on the migration of stimulating activityof the gelatin-binding domain of fibronectin and IGDS (SEQ ID NO 2);

FIG. 4 is a graph showing the effects of pre-incubation of cells withsynthetic peptides on their subsequent migration in the native collagengel assay.

FIG. 5 is the primary amino acid structure of human fibronectin (SEQ IDNO 7);

FIG. 6 is a graph showing the modulation of the effect of GBD and theIGDS (SEQ ID NO 2) tetrapeptide on fibroblast migration into 3D collagenmatrices by cell density; and

FIG. 7 (top and bottom) are photomicrographs of the angiogenic activity15 of IGDS (SEQ ID NO 2) synthetic peptide and gelatin-binding domain offibronectin in the chick yolk sac membrane assay.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention will now be described in more detail with reference to thefollowing Figures and Examples in which:

FIG. 1 shows the effects of synthetic peptides and fibronectin domainson cell migration in the native collagen gel assay. Assays wereperformed as described in Materials and Methods in Example 1. Resultsare normalised by expressing them as “relative stimulation” ofmigration, this being calculated by dividing the percentage of cells inthe gel matrix for each experimental point by the control value obtainedin that particular experiment. Data are presented as the mean ±SD offive experiments. For clarity, the shaded area indicates the spread ofstandard deviations for data points not plotted with error bars. Upperpanel: results obtained with the indicated synthetic peptides. Lowerpanel: results obtained with native fibronectin (FN), and itscell-binding (Cell-BD) and gelatin-binding (Gel-BD) domains.

FIG. 2 shows the effects of synthetic peptides and fibronectin domainson cell migration in the transmembrane assay. Assays were performed asdescribed in Materials and Methods in Example 1. Results from fiveexperiments are presented as mean ±SD. Shaded area indicates the spreadof standard deviations for data points not plotted with error bars.Panel A: results obtained with IGDS (SEQ ID NO 2) and thegelatin-binding domain (Gel-BD) on membranes coated with native collagenand gelatin. Panel B: results obtained with RGDS (SEQ ID NO 6), nativefibronectin (FN), and its cell-binding domain (Cell-BD).

FIG. 3 shows the effect of different peptides on the migration ofstimulating activity of the gelatin-binding domain of fibronectin andIGDS (SEQ ID NO 2). The collagen gel assay was performed in the presenceof various combinations of the indicated peptides and the percentage ofcells present within the gel matrix measured after a four-day incubationperiod. Data are expressed as mean ±SD obtained in three experiments.Panel A: combinations of the gelatin-binding domain (Gel-BD) and IGDS(SEQ ID NO 2); Panel B: combinations of IGDS (SEQ ID NO 2) and RGDS (SEQID NO 6); Panel C: combinations of IGDS (SEQ ID NO 2) and thecell-binding domain (Cell-BD).

FIG. 4 shows the effect of pre-incubation of cells with syntheticpeptides on their subsequent migration in the native collagen gel assay.Confluent cells on plastic tissue culture dishes were washed 3× withserum-free MEM (SF-MEM) and then incubated for various times with theindicated concentrations of IGDS (SEQ ID NO 2) in SF-MEM. They were thentrypsinised and washed extensively by repeated (5×) cycles ofcentrifugation and resuspension in SF-MEM. The behaviour of thesepre-incubated cells was assessed in the collagen gel assay in theabsence of further IGDS (SEQ ID NO 2)(indicated as IGDS (SEQ ID NO 2)not in assay). These results were compared with the response of controlcells to IGDS (SEQ ID NO 2) present during the four-day duration of themigration assay (indicated as IGDS (SEQ ID NO 2) in assay). Data wereobtained from three experiments and are expressed as mean ±SD.

FIG. 5 shows the primary amino acid structure of human fibronectin.

Primary structure of Fibronectin

Ala A 100 Cys C 63 Asp D 126 Glu E 145 Phe F 54 Gly G 208 His H 51 Ile I121 Lys K 78 Leu L 136 Met M 27 Asn N 101 Pro P 195 Gln Q 133 Arg R 126Ser S 200 Thr T 268 Val V 200 Trp W 40 Tyr Y 105 Mol. Wt (calc.) = 273715 Residues = 2476

Structural sites

Signal peptide: 1–20

Propeptide: 21–31

Type I repeats: 52–96, 97–140, 141–185, 186–230, 231–272, 308–344,470–517, 518–560, 561–608, 2297–2341, 2342–2385, 2386–2428

Type II repeats: 345–404, 405–469

Type III repeats: 609–700, 719–809, 810–905, 906–995, 996–1085,1086–1172, 1173–1265, 1357–1447, 1448–1537, 1538–1631, 1632–1721,1812–1903-, 1904–1992, 1993–2082, 2203–2273

Alternatively spliced domains: 172–1811 (ED-A), 1266–1356 (ED-B),2083–2202 (IIICS)

Potential N-linked glycosylation sites: 430, 528, 542, 877, 1007, 1244,1291, 15 1904, 2199

O-Linked glycosylation site: 2155

Interchain disulphide bond residues: 2458, 2462

RGDD (SEQ ID NO 13) cell adhesion site: 1615–1618

(DAPS(SEQ ID NO 8) cell adhesion site: 1994–1998

LDV (SEQ ID NO 9) cell adhesion site: 2102–2104

REDV (SEQ ID NO 10) cell adhesion site: 2182–2185

Heparin-binding sites: 2028–2046 (FN-C/H I), 2068–2082 (FN-C/H II)

Factor Xllla transglutaminase cross-linking site: 34

FIG. 6 shows the modulation of the effect of GBD and the IGDS (SEQ ID NO2) tetrapeptide on fibroblast migration into 3D collagen matrices bycell density. Cells were plated at either confluent (conf) orsubconfluent (subconf) densities. Shaded area indicates range of controlvalues.

FIG. 7 shows the angiogenic activity of IGDS (SEQ ID NO 2) syntheticpeptide and gelatin-binding domain of fibronectin in the chick yolk sacmembrane assay. Dried methylcellulose pellets containing the testsamples and control pellets (lacking the test samples) were placed onthe yolk sac membrane of 6-day chick embryos, as described in Materialsand Methods in Example 1. The elicited reaction was checked after sixhours and assessed after 24 hr by observation of living embryos with astereomicroscope. A. negative angiogenic response (in this case,elicited by control pellet); B. typical positive angiogenic response (inthis case, elicited by pellet containing 1.0 μg IGDS (SEW ID NO 2)).After assessment at 24 hr, selected membranes were fixed in 2.5% EMgrade glutaraldehyde in 0.2 M phosphate buffer, pH 7.4. Thesepreparations were then stained with 1% toluidine blue and photographedusing a Leica DM LB microscope. C. appearance of fixed and stainedpositive angiogenic response (in this case, elicited by pelletcontaining 1.0 μg of the gelatin-binding domain). Bar=250 μm.

EXAMPLE 1 Migration Stimulating Activity of the IGD (SEQ ID NO 1) AminoAcid Motif Summary

The gelatin-binding domain of fibronectin stimulates fibroblastmigration into matrices of native type I collagen, but is devoid of suchactivity with cells adherent to a denatured collagen substratum. We nowdemonstrate the IGD (SEQ ID NO 1) motif, present at two sites within thegelatin-binding domain, displays the same substratum-dependent activity.Micromolar concentrations of IDG-containing synthetic peptidesstimulated fibroblast migration into native (but not denatured) collagensubstrata in the following activity order: IGDS (SEQ ID NO 2)>IGDQ (SEQID NO 3)>IGD (SEQ ID NO 1). The related RGDS (SEQ ID NO 6) peptide didnot affect cell migration on its own and inhibited the bioactivity ofIGDS (SEQ ID NO 2) in a dose-dependent fashion. Cells pre-incubated withIGDS (SEQ ID NO 2) displayed a persistent stimulation of cell migrationwhen assayed in the absence of further IGDS. This feature of IGDS (SEQID NO 2) bioactivity provided a means to study the early events of IGDS(SEQ ID NO 2) action (for example, receptor ligation and post-ligationsignalling) separately from the late events resulting in the persistentstimulation of cell migration. Accordingly, experiments in which cellswere incubated with IGDS (SEQ ID NO 2) and inhibitors in varioustemporal combinations indicated that (a) both early and late events ofIGDS (SEQ ID NO 2) action were effectively inhibited by RGDS, as well asfunction-blocking antibodies to integrin subunits (β₁ and β₃) andheterodimer α_(v)β₃, (b) neutralising antibodies to the classicfibronectin-binding α₅β₁, integrin were without effect, and (c)inhibition of tyrosine kinase activity blocked early events of IGDS (SEQID NO 2) action, inhibition of MAP kinase kinase blocked both early andlate events, whilst inhibition of PKA only affected late events. In vivostudies further indicated that IGDS (SEQ ID NO 2) synthetic peptideelicited an angiogenic response in the chick yolk sac membrane; incontrast, RGDS (SEQ ID NO 6) and RGES (SEQ ID NO 11) peptides wereinactive under the same experimental conditions. The expression ofbiological activity by IDG (SEQ ID NO 1) synthetic peptides (both invitro and in vivo) stands in marked contrast to the inactivity of theirwell-studied RGDS (SEQ ID NO 6) counterparts and opens the possibilityof developing a novel family of clinically relevant agents.

Materials and Methods

Chemicals. The synthetic peptides were prepared to greater than 99%purity in the Department of Biochemistry, University of Dundee.Fibronectin and its cell-binding and gelatin-binding domains werepurified as previously described⁷. Monoclonal antibodies to the integrinsubunits α2 (cat. no. MCA743) and β1 (MCA1188) were supplied by Serotec(Oxford, UK); antibodies to β3 (MAB1957), αvβ3 (MAB1976) and α5β1 (MAB1969) were supplied by Chemicon (Harrow, UK); antibody to α5β1 (M0604)was supplied by Dako (High Wycombe, UK). Genistein (cat. no. 34583-Q)and PD98059 (cat. no. 178278-Q) were purchased from Calbiochem,Nottingham. PKA inhibitor peptide (cat. no. P6062) was purchased fromSigma Chemical Co (Poole, Dorset, UK).

Cells. Experiments were performed with two lines of human skinfibroblasts (SK319 and FSF44, between passage 10–18) shown to be free ofmycoplasmal contamination by staining with Hoechst 33256. Identicalresults were obtained with both lines and these cells are consequentlynot individually identified in the Figures. Stock cultures weremaintained in Eagle's Minimal Essential (MEM), as previously described⁷.

Migration Assays. In the collagen gel assay, pre-formed 2 ml gels wereoverlaid with 1 ml of serum-free MEM (controls) or serum-free MEMcontaining the requisite concentration of effector molecule to give thedesired final concentration. Trypsinised fibroblasts were suspended inserum-free MEM to give an inoculum containing 2×10⁵ cells/ml and 1 ml ofthis was plated onto replicate control and test gels. After a 4-dayincubation period at 37° C., the cells on the surface and within the 3Dmatrix of the gel were counted in 15 randomly selected fields bymicroscopic observation and these data used to calculate the percentageof total cells present within the gel matrix⁶.

Polycarbonate membranes used in the transmembrane assay were immersed inan aqueous solution containing 10 μg/ml of either native type I collagenor heat-denatured type I collagen (gelatin) overnight at 37° C. and thenair-dried. Assays were performed as previously described⁷.

Chick yolk sac membrane angiogenesis assay. The assay was performedessentially as described by Gush et al (1990) J. Med. Engineer. Tech.14, 205–209. Accordingly, four-day-old fertilised eggs were cracked in atumbler, covered with a Petri dish and incubated at 37° C. Two dayslater dried methylcellulose pellets containing the test samples andcontrol pellets (lacking the test samples) were placed on the yolk sacmembrane. The elicited angiogenic reaction was assessed after 24 hr byobservation of living embryos with a stereomicroscope. Selectedmembranes were fixed in 2.5% EM grade glutaraldehyde in 0.2 M phosphatebuffer (pH 7.4), dissected, stained with 1% toluidine blue and mountedon glass slides for photomicroscopy.

Results and Discussion

Inspection of the amino acid sequence of the gelatin-binding domainrevealed that it contains two IGD (SEQ ID NO 1) motifs located in theseventh and ninth type I repeat modules, respectively. This is ofparticular interest, as the IGD (SEQ ID NO 1) motif is a highlyconserved feature of the type I module⁸ and its location at the apex ofthe main type I loop is homologous to that of the RGD (SEQ ID NO 4)motif in the tenth type III repeat⁹. Relevant IGD-containing syntheticpeptides were synthesised and their effect on the migration of humandermal fibroblasts examined in the collagen gel assay. Resultssummarised in FIG. 1A indicate that IGDS (SEQ ID NO 2)(as present in theninth type I module), IGDQ (SEQ ID NO 3)(as present in the seventh typeI module) and IGD (SEQ ID NO 1) stimulated cell migration into nativetype I collagen gels in a dose-dependent fashion. Significantbioactivity was expressed by IGDS (SEQ ID NO 2) and IGDQ (SEQ ID NO 3)at a concentration of 0.1 μM, whilst comparable activity was firstmanifest by IGD (SEQ ID NO 1) at 10–100 μM. IGDS (SEQ ID NO 2) produceda bell-shaped dose-response; this was not obtained with either IGDQ (SEQID NO 3) or IGD (SEQ ID NO 1) within the concentration range examined.The structurally related RGDS (SEQ ID NO 6) tetrapeptide was devoid ofmigration stimulating activity in the native collagen gel assay.Comparative results obtained with purified, proteolytically-generated,fibronectin fragments (FIG. 1B) confirmed our previous observations thatthe gelatin-binding domain exhibits significant migration stimulatingactivity, whilst native fibronectin and its purified cell-binding domainare inactive when tested within the same concentration range⁷.Comparison of data presented in FIGS. IA and B further indicate that themicromolar concentration range of IGD (SEQ ID NO 1)-containing syntheticpeptides required to induce a stimulation of cell migration is manyorders of magnitude greater than the corresponding femtomolarconcentration range of the larger gelatin-binding domain.

These observations suggest the involvement of other amino acid motifswithin the gelatin-binding domain in facilitating recognition and/orbinding of IGD (SEQ ID NO 1) to its putative cell surface receptor. Inthis context, Aota et al¹⁰ reported that the PHSRN (SEQ ID NO 12)sequence in the ninth type III module of the cell binding domain is sucha “synergistic” motif for RGD-dependent biological activity.

The effects of a native and denatured type I collagen substratum on themigration stimulating activity of IGD-containing synthetic peptides wasassessed in the transmembrane assay. Data presented in FIG. 2A indicatethat IGDS (SEQ ID NO 2) stimulated cell migration through membranescoated with native collagen, but was devoid of activity ongelatin-coated membranes. Similar results were obtained with IGDQ (SEQID NO 3) and IGD (SEQ ID NO 1)(data not shown). These observationsindicate that the bioactivity of IGD-containing synthetic peptides is(a) dependent upon cell attachment to a native collagen substratum, and(b) resembles that of the larger gelatin-binding domain in which it iscontained in terms of this criterion. The mechanism responsible for thesubstratum-dependent nature of IGD (SEQ ID NO 1) activity remains to bedetermined. In this regard, it may be relevant that cellular adhesion toa native collagen substratum specifically affects a number of cellularprocesses of potential relevance to the modulation of cell migration(for example, phosphorylation of pp125FAK and activation ofPKC-ζ)^(11,12).

Data presented in FIG. 2B confirm that native fibronectin and itspurified cell binding domain stimulate cell migration through membranescoated with gelatin (as reported in previous studies) and that the RGDS(SEQ ID NO 6) synthetic peptide is inactive on both collagen- andgelatin-coated membranes.

The possible mechanistic relationship between IGDS (SEQ ID NO 2) and thegelatin-binding domain in which it is contained was further examined byco-incubating cells with suboptimal concentrations of each. The resultspresented in FIG. 3A indicate that these two peptides exerted anadditive effect upon cell migration, consistent with the hypothesis thatthe IGD (SEQ ID NO 1) motif within the gelatin-binding domain is indeedresponsible for its stimulation of cell migration. This additive effectwas particularly apparent at 0.1 fM GBD and 0.01 μM (10⁷ fM) IGDS, whichwere each inactive when present on their own, but active in combination.

Possible mechanistic interactions between IGDS (SEQ ID NO 2) and RGDS(SEQ ID NO 6) were studied in similar co-incubation experiments. Resultspresented in FIG. 3B indicate that RGDS (SEQ ID NO 6) effectivelyinhibited the migration stimulating activity of IGDS (SEQ ID NO 2). Thecell-binding domain of fibronectin (which contains the RGDS (SEQ ID NO6) motif also inhibited IGDS (SEQ ID NO 2) activity (FIG. 3C). Asexpected, the inhibition of IGDS (SEQ ID NO 2) activity was achieved atconsiderably lower (nanomolar) concentrations of the cell-binding domaincompared to the pmolar concentrations required of RGDS (SEQ ID NO 6).The RGES (SEQ ID NO 11) synthetic peptide had no effect on IGDS (SEQ IDNO 2) migration stimulating activity when tested at the sameconcentration range as RGDS (SEQ ID NO 6)(data not shown).

A number of biological activities of fibronectin are “cryptic” in thesense that they are displayed by fibronectin proteolytic fragments, butnot by the intact molecule^(5,13). Fukai et al¹⁴ have demonstrated thatthe expression of these cryptic activities require either thedenaturation of native fibronectin and/or its limited proteolyticdegradation into functional domains in order to become manifest. Theseauthors suggest that relaxation of steric hindrance may be responsiblefor the unmasking of latent biological activity by these procedures. Theinhibition of IGDS (SEQ ID NO 2)-induced cell migration by the RGDS (SEQID NO 6) amino acid motif may provide an additional mechanism for theapparent lack of IGDS (SEQ ID NO 2) activity in native fibronectin (FIG.1 B).

We have previously reported that cells pre-incubated for 24 hr with thegelatin-binding domain of fibronectin displayed elevated migratoryactivity when subsequently plated on native collagen gels in itsabsence⁷. Data presented in FIG. 4 indicate that the effect of IGDS (SEQID NO 2) on cell migration is similarly persistent, exhibiting adependence upon both the time of pre-incubation and peptideconcentration. This elevated migratory behaviour is still manifest bypre-incubated cells following 1–2 passages in vitro (data not shown).

The persistence of IGDS (SEQ ID NO 2)-bioactivity suggests that its modeof action involves a series of early events which are dependent upon thepresence of IGDS (SEQ ID NO 2)(such as receptor ligation andpost-ligation signalling) and later events, which no longer require thepresence of IGDS (SEQ ID NO 2) and ultimately result in stabilisation ofa persistent migratory phenotype. The specific effects of potentialinhibitory molecules on such early and late events were examined byusing the following experimental protocol: (a) pre-incubating cells withIGDS (SEQ ID NO 2) and inhibitor and then assaying these treated cellsin the absence of both IGDS (SEQ ID NO 2) and inhibitor (note: thisprotocol provides data concerning the effects of inhibitor on the earlyevents mediating IGDS (SEQ ID NO 2) activity), (b) pre-incubating cellswith neither IGDS (SEQ ID NO 2) nor inhibitor and assaying them in thepresence of both (the effects of inhibitor on both early and lateevents), (c) pre-incubating cells with IGDS (SEQ ID NO 2) alone and thenassaying them in the presence of inhibitor only (the effects ofinhibitor on late events), and finally, (d) pre-incubating cells withinhibitor alone and then assaying them in the presence of IGDS (SEQ IDNO 2) to provide control information regarding possibly persistenteffects of inhibitor which would confound data interpretation. Datapresented in Table IA are concerned with the effects of the syntheticRGDS (SEQ ID NO 6) and RGES (SEQ ID NO 5). Our results indicate thatRGDS (SEQ ID NO 6) inhibited both IGDS (SEQ ID NO 2) cell signalling andsubsequent cell migration. RGES (SEQ ID NO 11) was inactive under allexperimental conditions. Control data (protocol “d”) indicated that bothpeptides had no persistent effect on IGDS (SEQ ID NO 2) activity; all ofthe other inhibitors examined were similarly devoid of such potentiallyconfounding activity (data not shown).

The inhibitory effects of RGDS (SEQ ID NO 6) on IGDS-induced cellmigration may occur by competition for receptor ligation. In order toobtain data relevant to this possibility, we employed the abovepre-incubation protocols to examine the effects of neutralisingantibodies to several integrins expressed by human dermal fibroblasts invitro¹⁵ Our data indicate that the monoclonal antibody recognising thea2 integrin subunit inhibited cell migration induced by IGDS (SEQ ID NO2), but did not affect initial IGDS (SEQ ID NO 2) signalling (Table 3B).This observation is consistent with the role of α2β1 in mediating cellattachment to collagen and its involvement in supporting cell migrationon this substratum¹⁶. Antibodies to the αvβ3 heterodimer, as well as tothe integrin subunits α1 and β3, were found to block both the initialevents of IGDS (SEQ ID NO 2) signalling and subsequent cell migration.The αvβ3 heterodimer recognises the RGDS (SEQ ID NO 6) motif, whilst theβ₁ and β₃ subunits are present in several integrin heterodimers whichalso recognise RGDS^(15,17). Several previous studies have implicatedthese integrins in the mediation of cell migration^(16,18). In thiscontext, it should be noted that the α_(v)β₃ heterodimer also binds toexposed RGD (SEQ ID NO 4) sites in denatured (but not native)collagen¹⁹; this differential ligation of denatured and native type Icollagen by α_(v)β₃ may contribute to the substratum-dependent nature offibroblast migratory response to IGD-containing peptides reported here.Two antibodies to the “classic” fibronectin-binding α₅β₁ integrin had noeffect on IGDS-induced cell migration (Table 3B). These observations areconsistent with previous reports suggesting that integrin α₅β₁preferentially mediates cell adhesion rather than migration²⁰. Recentstudies have underscored the interplay between substratum, ligandconcentration and integrin function in the control of cellmigration^(21,22); these complex factors will need to be taken intoaccount in identifying the precise integrin receptors involved in IGD(SEQ ID NO 1) ligation and the post-ligation events leading to theresultant substratum-dependent biological activity.

Results obtained with signal transduction inhibitors indicate that thetyrosine kinase inhibitor Genistein selectively blocked IGDS (SEQ ID NO2)-induced cell signalling, but did not affect cell migration (Table1C). This finding is consistent with the role of focaladhesion-associated tyrosine kinases (such as pp125FAK) in mediatingintegrin signal transduction²³. The MAP kinase kinase inhibitor PD98059blocked both signal transduction and cell migration, in keeping with thepreviously reported activation of the MAP kinase cascade by integrinligation²⁴. In contrast, the PKA inhibitor blocked cell migration, butdid not appear to affect initial IGDS (SEQ ID NO 2)-dependent events.

In addition to RGD, other amino acid sequences in fibronectin have beenreported to mediate cell adhesion and migration; these include LDV (SEQID NO 9), REDV (SEQ ID NO 9) and IDAPS (SEQ ID NO 8)¹⁷. All these motifsresemble RGD (SEQ ID NO 4) in that migration stimulating activity is notretained by the respective soluble synthetic peptides. Themigration-stimulating activity of IGD-containing synthetic peptidesappears to be unique in this sense. Although biological activity has notpreviously been ascribed to the conserved IGD (SEQ ID NO 1) motif infibronectin, previous studies have implicated the ninth type I repeat(which contains the IGDS (SEQ ID NO 2) sequence in the assembly of anextracellular fibronectin matrix²⁵. The data presented here may berelevant in this context and suggest several integrins which mayfunction in IGDS (SEQ ID NO 2) ligation.

The migration inhibiting activity of RGD-containing peptides has anumber of potential clinical applications^(26,27). Structure-functionstudies have indicated that conservative and non-conservative amino acidsubstitutions, tandem amino acid extensions and cyclicisationsignificantly modulate the biological activity of the RGD (SEQ ID NO 4)motif in these situations^(28,29). The converse migration-stimulatingactivity of IGD (SEQ ID NO 1)-containing synthetic peptides may providean analogous platform for developing a new family of therapeutic agentswhich promote cell migration in clinically relevant conditions, such asimpaired wound healing.

EXAMPLE 2 Angiogenic Response in a Rat Wound Healing Model

The IGDS (SEQ ID NO 2) peptide has been shown to stimulate fibroblastmigration and elicit and angiogenic response in a rat wound healingmodel. In this experimental system, 1 cm² pieces of porcine dermalcollagen films impregnated with either control medium or mediumcontaining the indicated concentration of test substance were implantedsubcutaneously into rats. The animals were sacrificed 28 days later andthe removed collagen implants fixed and sectioned for image analysis.The following data were obtained, indicating that IGDS (SEQ ID NO 2)stimulated both fibroblast migration into the collagen film and anangiogenic response.

Vessels (per field) Fibroblasts (% field) Control 15.3 ± 5.5  8.3 ± 6.7IGDS (1 μg/ml) 26.7 ± 6.4 18.2 ± 9.4 p < 0.01 p < 0.01

TABLE 3 Effects of various inhibitors on early (receptor ligation andsignalling) and late persistent stimulation of cell migration) aspectsof IGDS (SEQ ID NO 2) activity. A: synthetic peptides Pre- InhibitoryPeptide incubation In assay % activity (μ molar) IGDS Peptide IGDSpeptide inhibition yes(+) no(−) RGDS − − + + 92.3 ± 2.1 early events: +(10.0) + + − − 96.2 ± 4.8 late events: + + − − + 96.5 ± 5.7 RGES − − + + 1.2 ± 2.1 early events: − (10.0) + + − − −1.0 ± 3.0 late events: − + −− +  0.3 ± 2.8 B: integrin antibodies Pre- incubation In assayInhibitory Antibody anti- anti- % activity (μg/ml) IGDS body IGDS bodyinhibition yes(+) no(−) α₂ − − + + 92.9 ± 6.1 early events: − (10.0) + +− − −5.7 ± 2.0 late events: + + − − + 97.4 ± 8.9 β₁ − − + + 96.1 ± 2.1early events: + (10.0) + + − − 92.8 ± 3.9 late events: + + − − + 99.1 ±5.5 β₃ − − + + 94.0 ± 4.2 Early events: + (10.0) + + − − 98.0 ± 1.0 Lateevents: + + − − + 96.7 ± 3.6 α₅β₁ − − + +  4.1 ± 3.3 Early events: −(10.0) + + − −  3.6 ± 2.8 Late events: − + − − + −0.1 ± 2.6 α_(v)β₃ −− + + 97.9 ± 4.7 Early events: + (10.0) + + − − 88.0 ± 3.3 Lateevents: + + − − +  68.9 ± 12.0 C: signal transduction inhibitors Pre-incubation In assay Inhibitory Inhib- Inhib- % activity Inhibitor IGDSitor IGDS body itor yes(+) no(−) Genistein − − + + 94.1 ± 6.5 earlyevents: + (10 μg/ml) + + − − 96.7 ± 4.0 late events: − + − − +  0.9 ±2.9 PD98059 − − + + 99.8 ± 3.8 early events: + (2.0 μM) + + − − 90.5 ±3.1 late events: + + − − + 39.4 ± 5.0 PKA inhib − − + + 90.0 ± 6.4 earlyevents: − (5 nM) + + − −  6.9 ± 5.7 late events: + + − − + 99.5 ± 4.2

TABLE 4 Angiogenic Activities of Synthetic Peptides and Gelatin-bindingDomain of Fibronectin. The angiogenic activities of the indicated testcompounds were ascertained in the chick yolk sac assay, as previouslydescribed in Gush et al (1990) J. Med. Engineer. Tech. 14, 205–209. SeeLegend FIG. 7 for further details. Angiogenic Activity Positiveresponses Compound Concentration (ng/pellet) (%) Control — 2/28 (7) IGDS5 3/6 (50) 50 7/11 (64) 250 10/13 (77) 1000 9/14 (64) 3000 7/9 (78)Gelatin-binding 5 0/4 (0) domain 50 3/10 (30) 250 7/8 (87) 1000 9/12(75) 3000 6/6 (100) RGDS 3000 1/11 (9) RGES 3000 1/10 (10)

REFERENCES

The following references are hereby incorporated by reference.

-   1. Zardi, L. et al (1985) Eur. J. Biochem. 146, 571–579.-   2. Postlethwaite, A. E. et al (1981) J. Exp. Med. 153, 494–499.-   3. Albini, A. et al (1987) J. Cell. Biol. 105, 1867–1872.-   4. Akiyama, S. K. & Yamada, K. M. (1985) J. Biol. Chem. 260,    10402–10405.-   5. Clark, R. A. F. et al (1988) J. Biol. Chem. 263, 12115–12123.-   6. Schor, S. L. (1980) J. Cell Sci. 41, 159–175.-   7. Schor, S. L. et al (1996) J. Cell Sci. 109, 2581–2590.-   8. Hynes, R. O. (1990) Fibronectins pp 132–135, Springer-Verlag: New    York.-   9. Main, A. L. et al (1992) Cell. 71, 671–678.-   10. Aota, S. et al (1994) J. Biol. Chem. 269, 24756–24761.-   11. Roekel, D. & Krieg, T. (1994) Exp. Cell Res. 211, 42–48.-   12. Xu, J. & Clark, R. A. F. (1997) J. Cell Biol. 136, 473–483.-   13. Fukai, F. et al (1993) Biochem. 32, 5746–5751.-   14. Fukai, F. et al (1995) Biochem. 34, 11453–11459.-   15. Gailit, J. & Clark, R. A. F. (1996) J. Cell Biol. 106, 102–108.-   16. Yamada, K. M. et al (1990) Cancer Res. 50, 4485–4496.-   17. Yamada, K. M. (1991) J. Biol. Chem. 266, 12809–12812.-   18. Tooney, P. A. (1993) Immunol. Cell Biol. 71, 131–139.-   19. Davis, G. E. (1992) Biochem. Biophys. Res. Commun. 182,    1025–1031.-   20. Chan, B. M. et al (1992) Cell. 68, 1051–1060.-   21. Schwartz, M. A. et al (1995) Annu. Rev. Cell Dev. Biol. 11,    549–599.-   22. Palecek, S. P. et al (1997) Nature 385, 537–540.-   23. Richardson, A. & Parson, J. T. (1995) BioEssays 17, 229–236.-   24. Chen, Q. et al (1994) J. Biol. Chem. 269, 26602–26605.-   25. Chernousov, M. A. et al (1991) J. Biol. Chem. 266, 10851–10858.-   26. Humphries, M. J. et al (1994) Exp. Opin. Ther. Patents 4,    227–235.-   27. Pierschbacher, M. D. et al (1994) J. Cell Biochem. 56, 150–154.-   28. Yamada, K. M. & Kennedy, D. W. (1985) J. Cell Biochem. 28,    99–104.-   29. Pierschbacher, M. D. & Ruoslahti, E. (1987) J. Biol. Chem. 262,    17294–17298.-   30. Hunt, T. K. Wound Healing and Infection: Theory and Surgical    Practice Appleton-Century-Crofts: New York (1980).-   31. Britsch, S., Christ, B. & Jacob, H. J. “The influence of    cell-matrix interactions on the development of quail chorioallantoic    vascular system” Anal. Embryol. 180, 479–484.

1. A composition comprising a compound consisting of a sequence selectedfrom the group consisting of Ile-Gly-Asp (IGD) (SEQ ID NO:1),Ile-Gly-Asp-Ser (IGDS) (SEQ ID NO:2), Ile-Gly-Asp-Gln (IGDQ) (SEQ IDNO:3), and a non-peptide or peptide mimic thereof, wherein the compoundhas cell migration increasing activity; wherein if the sequence is apeptide mimic, the Ile is replaced with an amino acid selected from thegroup consisting of Val, Leu, Phe, Trp, and Tyr; or the Asp is replacedwith Glu, or the Gly is replaced with Ala.
 2. The composition accordingto claim 1, wherein the sequence is a peptide mimic and wherein the Ileis replaced with an amino acid selected from the group consisting ofVal, Leu, Phe, Trp, and Tyr.
 3. The composition according to claim 1,wherein the sequence is a peptide mimic and wherein the Asp is replacedwith Glu, or wherein the Gly is replaced with Ala.
 4. The compositionaccording to claim 1 wherein the amino acids isoleucine (I), glycine(G), aspartic acid (D), serine (S), glutamine (Q), valine (V), leucine(L), phenylalanine (F), tryptophan (W), tyrosine (Y), glutamic acid (E),or alanine (A) within the compound are in the L-configuration.
 5. Apharmaceutical composition comprising the compound according to claim 1and a pharmaceutically acceptable carrier.
 6. A method of modulatingcell migration, the method comprising administering to the site wherecell migration is desired a therapeutically effective amount of acompound with a relative molecular mass of less than 15,000 comprisingthe peptide Ile-Gly-Asp (IGD) (SEQ ID NO:1) or a non-peptide or peptidemimic thereof with cell migration increasing activity, wherein if thesequence is a peptide mimic, the Ile is replaced with an amino acidselected from the group consisting of Val, Leu Phe, Trp, and Tyr, or theAsp is replaced with Glu, or the Gly is replaced with Ala.
 7. The methodaccording to claim 6, wherein the compound is a peptide mimic andwherein the Ile is replaced with an amino acid selected from the groupconsisting of Val, Leu, Phe, Trp, and Tyr, or wherein the Asp isreplaced with Glu, or wherein the Gly is replaced with Ala.
 8. Themethod according to claim 6 wherein the cell is a fibroblast.
 9. Themethod according to claim 6 wherein the cell migration site is in ananimal body.
 10. The method according to claim 6 wherein the cellmigration site is in a mammalian body.
 11. The method according to claim8 wherein the cell migration site is in a human body.
 12. The methodaccording to claim 6 for increasing cell migration.
 13. A compound withcell migration increasing activity and a molecular mass of less than15,000 comprising a plurality of sequences selected from the groupconsisting of Ile-Gly-Asp (IGD) (SEQ ID NO:1), Ile-Gly-Asp-Ser (IGDS)(SEQ ID NO:2), Ile-Gly-Asp-Gln (IGDQ) (SEQ ID NO:3), and a non-peptideor peptide mimic thereof, wherein if the sequence is a peptide mimic,the Ile is replaced with an amino acid selected from the groupconsisting of Val, Leu, Phe, Trp, and Tyr; or the Asp is replaced withGlu, or the Gly is replaced with Ala.
 14. The composition of claim 2,wherein the Ile in the peptide mimic is replaced with an amino acidselected from the group consisting of Val, Leu, Phe, and Tyr.
 15. Thecomposition of claim 2, wherein the Ile in the peptide mimic is replacedwith an amino acid selected from the group consisting of Val and Leu.16. A composition comprising a compound consisting of a sequenceselected from the group consisting of Ile-Gly-Asp (IGD) (SEQ ID NO:1),Ile-Gly-Asp-Ser (IGDS) (SEQ ID NO:2), Ile-Gly-Asp-Gln (IGDQ) (SEQ IDNO:3), and a non-peptide or peptide mimic thereof, wherein the compoundhas cell migration increasing activity; and wherein if the sequence is apeptide mimetic; the Asp is replaced with Glu, or the Gly is replacedwith Ala.
 17. A composition with cell migration increasing activity anda molecular mass of less than 15,000 comprising more than one compoundselected from the group consisting of Ile-Gly-Asp (IGD) (SEQ ID NO:1),Ile-Gly-Asp-Ser (IGDS) (SEQ ID NO:2), Ile-Gly-Asp-Gln (IGDQ) (SEQ IDNO:3), a non-peptide and peptide mimic thereof, wherein the compound hascell migration increasing activity, wherein if the sequence is a peptidemimic, the Ile is replaced with an amino acid selected from the groupconsisting of Val, Leu, Phe, Trp, and Tyr, or the Asp is replaced withGlu, or the Gly is replaced with Ala.
 18. The peptide of claim 13,wherein the compound comprises a plurality of sequence Ile-Gly-Asp (IGD)(SEQ ID NO:1).